International Journal of Bioinformatics and Biomedical Engineering Vol. 1, No. 2, 2015, pp. 101-106 http://www.aiscience.org/journal/ijbbe

Elimination of Dengue by Control of Aedes Vector Mosquitoes (Diptera: Culicidae) Utilizing (Copepoda: )

Muhammad Sarwar *

Nuclear Institute for Agriculture & Biology, Faisalabad, Punjab, Pakistan

Abstract This paper reports on the information and result of long-term laboratory and field studies on copepods (Copepoda: Cyclopidae) as predators for control inhabiting in tropic and subtropic environments. Mosquitoes have long been vectors of numerous diseases that affect human health and well-being in many parts of the world. Reducing the use of pesticides against insect vectors is one of the big demands of the society because public has always been against the heavy use of insecticides. Copepods are natural and tiny shrimp-like with a hearty appetite for feeding on mosquito larvae in water holding areas. The copepods thrive in fresh and marine water, and are valuable tool in battling mosquitoes in artificial containers, roadside ditches, small water pools, clogged downspouts and other wet areas that can breed plenty of mosquitoes. These are especially helpful tools in fighting mosquitoes near public places, where use of certain pesticides is restricted. Copepods are relatively easy to culture, maintain and deliver to the target areas, but getting the cultures started requires some effort and time. Copepods are more efficient predator of younger than of older larvae of mosquito and predation drops considerably for 4 days and older larvae. Copepods though prefer to prey on younger larvae, yet also increasingly attack on older larvae as greater predator densities reduce the supply of younger ones. Recent trials show that each might destroy forty larvae per day, cutting Aedes breeding by 99 to 100 percent and in practice these can best be used where most of the local mosquito problem is due to Aedes breeding locations. Once established, copepods are able to survive and reproduce well to maintain viable populations under a wide variety of field conditions. The use of these biological control agents is only one small part of a statewide integrated approach to mosquito control, and not a replacement for long-established procedures. The future additional research can build up new ways of producing and keeping alive copepods that might target specifically dengue vector mosquitoes, but few realistic problems still need to overcome.

Keywords Dengue, Copepods, , Mosquito Control, Biological Control

Received: May 24, 2015 / Accepted: June 5, 2015 / Published online: July 23, 2015 @ 2015 The Authors. Published by American Institute of Science. This Open Access article is under the CC BY-NC license. http://creativecommons.org/licenses/by-nc/4.0/

1. Introduction

Throughout the world, mosquitoes transmitted diseases such mosquito is a significant opportunity for the future control of as dengue, malaria and yellow fever are serious threat to vectors borne diseases, principally in view of existing limits on use of pesticides, continuing problems of resistance to human health. Moreover, mosquitoes are also severe chemicals and management of habitat. The public is normally annoyance with noteworthy interference on the economy of not always aware of some of the things protecting them, state and quality of public life. Biological control of particularly a variety of biological control agents making

* Corresponding author E-mail address: [email protected] 102 Muhammad Sarwar: Elimination of Dengue by Control of Aedes Vector Mosquitoes (Diptera: Culicidae) Utilizing Copepods (Copepoda: Cyclopidae) attractive to the countries. Quite a lot of species of copepods the ones that behave most aggressively, destroying larvae that (Copepoda: Cyclopidae), in various genera have shown come near even when these do not eat them. Recent tests assurance as biological control agents for control of with the copepod M. albidus in the laboratory and in field mosquitoes (Sarwar, 2014 a; 2014 b; 2014 c; 2014 d). conditions showed similar results, with almost a 90 percent The Cyclops is one of the most common genera of freshwater reduction in larvae. Tests have been done by adding them to copepods, belongs to Phylum Arthropoda, Subphylum water in containers, and so far, these have been fairly Crustacea, Subclass Copepoda, Order Cyclopoida, Family successful. Recent trials showed that these kill mostly first instar larvae of Aedes mosquitoes, but each copepod might Cyclopidae, and comprising over 400 species. Together with destroy forty larvae per day, cutting down Aedes breeding by other similar-sized non-copepod fresh-water , 99 to 100 percent. These are less effective with Anopheles especially Cladocera, these are commonly called water fleas having a single large eye, which may be either red or black in and least effective with Culex larvae. In practice, these would cyclops. Cyclops individuals may range from 0.5- 5 mm long best be used where most of the local mosquito problem is due to Aedes breeding in known locations, such as pools or water and are clearly divided into two sections. The broadly oval storage containers (Marten et al., 2000; Byer, 2004). front section comprises the head and the first five thoracic segments. The hind part is considerably slimmer and is made up of the sixth thoracic segments and the four legless pleonic 2. Biological Control of segments. Two caudal appendages project from the rear side Mosquito Larvae Using of body and have 5 pairs of legs. Owing to less than a couple millimeters length, these are barely visible to the naked eye, Copepods shaped like a teardrop, have long antennae and an Past studies on use of Cyclopoida have reported varying exoskeleton that is so thin that it is largely transparent. The results depending upon the copepods, mosquito species, long first antennae, 2 in number, are used by the males for weather, and the physical and biological milieu of the water gripping the females during mating. Afterwards, the female holding containers and areas. Six species of cyclopid carries the eggs in two small sacs on its body. The larvae or copepods have been tested for biological control of Aedes nauplii, are free-swimming and unsegmented. Cyclops has a albopictus larvae in discarded tires. Six to 8 weeks after cosmopolitan distribution in fresh water, but is less frequent introduction, Diacyclops navus, Acanthocyclops vernalls, in brackish water. It lives along the plant-covered banks of Mesocyclops ruttneri and Mesocyclops edax reduced the stagnant and slow-flowing bodies of water, where it feeds on number of A. albopictus larvae by 83, 90, 95 and.96%, small fragments of plant material, or carrion. It respectively. albidus and Mesocyclops swims with characteristic jerky movements. Cyclops has the longisetus are the most effective species. Six to 8 weeks after capacity to survive unsuitable conditions by forming a cloak introduction, M. albidus reduced A. albopictus larvae by 99%. of slime and average lifespan is about 3 months (Marten, Three months after introduction, M. albidus reduced A. 1986). albopictus larvae by 100%, and Mesocyclops longisetus Tiny copepods are aquatic crustaceans that are widespread in reduced A. albopictus by 99.8%. Macrocyclops albidus and both fresh and salt water habitats. The kinds of copepods M. longisetus are equally effective at eliminating Aedes proposed for mosquito control are referred to collectively as aegypti (L.), and A. triseriatus larvae (Marten, 1990 a). There cyclopoid copepods. These are voracious predators of is also evidence that natural populations of cyclopoids mosquito larvae and as such can be used to control mosquito eliminated Aedes and Anopheles larvae from ground water production from water holding areas. Particularly, these are habitats (Marten et al., 1989; Marten, 1989; 2000). Further natural enemies of the first and second instars (the smallest filed trials have been conducted to determine whether sizes) of mosquito larvae. The copepods attack and eat larvae, copepods can usefully destroy larval Stegomyia mosquitoes. but can also survive on other food sources in water, so they Copepods (Macrocyclops albidus ) have been released into can live for long periods even after these have eaten all the each of about 200 discarded tires arranged in 2 stacks of larvae, waiting for more progeny to hatch. Several species of about 100 tires, while a third stack remained untreated. copepods, particularly those in the genus Mesocyclops, have Larval A. albopictus that have been numerous in the treated shown promise as biological control agents for container- tires at the beginning of the experiment virtually disappeared breeding mosquitoes. A notable success in the use of within 2 months. Adults disappeared about 1 month later and copepods for reduction of disease vectors is that which remained scarce for at least another year. These predators, reports effective control of Aedes vectors of dengue with however, do not reduce the abundance of Culex salinarius , copepods of the genus Mesocyclops (Kay et al., 2002). Two which is another type of mosquito (Marten, 1990 b). kinds, Mesocyclops longisetus and Macrocyclops albidus, are In laboratory tests, four different strains of Mesocyclops International Journal of Bioinformatics and Biomedical Engineering Vol. 1, No. 2, 2015, pp. 101-106 103

aspericornis (Daday) showed potential as biological control appeared to preferably prey on Aedes larvae compared with agents of A. aegypti mosquito larvae, but have not been as Culex. Operationally, the use of M. longisetus as a tool for effective against Anopheles or Culex. In contrast, the larger control of container-inhabiting mosquitoes in urban or Mesocyclops longisetus (Thiebaud), killed 100% of A. suburban settings proved to be relatively inexpensive, aegypti and Anopheles farauti (Laveran) at larval densities of required little labor for colony maintenance, is easily 200 per liter and (Say) at 25 per liter. transported and smoothly applied to the target areas In cage simulations with A. aegypti and Mesocyclops , both (Soumare and Cilek, 2011). copepod species eliminated all immatures in earthenware As the most successful type of invertebrate used for mosquito pots by week 3. Owing to the lack of replacement, all A. control is the cyclopoid copepods, so most notably aegypti adults subsequently died by week 8 or 9. Although Mesocyclops that are one of the most numerous multicellular both M. longisetus and M. aspericornis showed maximum organisms on earth that can be found in many geographical reproductive potential at 25°C, yet breeding occurred from locations, and therefore the use of copepods for mosquito 20 to 35°C (Kay et al., 1992). A field trial used copepods in control does not require exotic introductions. Because of water tanks, tires and vases to control A. aegypti. It has been their small size, copepods mainly kill the first instar larvae, found that the most effective control is in the cemetery vases, and these prefer Aedes larvae over Anopheles and Culex with 67.5% reduction over the 3 month study period larvae. Copepods can live for 1-2 months, are quite hardy and (Gorrochotegui-Escalante et al., 1998). Copepods have also they self-replicate readily. Because these eat a variety of been used to successfully control A. aegypti populations in aquatic prey, they can maintain populations in water storage Argentina (Marti et al., 2004) and A. albopictus population in containers even if mosquitoes are not found (Marten and Japan has been effectively controlled by Macrocyclops Reid, 2007; DeRoa et al., 2002). These can also be easily copepods (Dieng et al., 2002). moved from one container to other container habitats; The cyclopoid copepod Macrocyclops albidus (Jurine) has therefore, they offer the potential of sustainable mosquito been tested as a potential biological control agent of control. Furthermore, copepods can be easily and cheaply mosquitoes in laboratory microcosms, in controlled field mass produced and transported, even under field conditions conditions, and in a field experiment using discarded tires. where they are required. Nam et al., (2000) used a method The predator is highly efficient in controlling mosquitoes in using plastic garbage bins in which thousands of copepods all three settings, reaching close to 90% reduction in larval could be produced in just 3 weeks. They then transported survival under field conditions and exceeding the these copepods to the various field locations using hollowed recommended predation rates for effective mosquito control polystyrene blocks. in laboratory experiments (effective on 1-4 day old larvae). The major success story for the use of copepods against Alternate food and habitat structure significantly influenced dengue vectors comes from a study carried out using the predation rates on mosquito larvae. Once established, the Mesocyclops. As the primary control measure, copepod has copepod is able to maintain reproducing populations in the been able to reduce A. aegypti levels to 0-0.3% of baseline field for the duration of the experiments (Jorge et al., 2004). estimates and A. albopictus to 0-14.1% of baseline levels (Kay The cyclopoid crustacean M. longisetus has been evaluated et al., 2002). The authors report that Aedes mosquitoes have for its predatory potential to reduce container-inhabiting been eliminated from several study communes following mosquitoes in backyards. A. albopictus , A. country-wide control program, and no dengue has been triseriatus and Culex quinquefasciatus have been the detectable in the three treated areas, but dengue transmission is predominant species collected from containers. At an initial still evident in the control areas (Nam et al., 2005). In his way, inoculation rate of 120 copepods per container, M. longisetus dengue has not been reported in the treated areas for years but populations eliminated resident mosquito larvae for a dengue transmission remained in the untreated areas (Kay and minimum of 14 wks in 30 liter plastic buckets, up to 29 wks Nam, 2005). Use of copepod Mesocyclops is also proving to in 0.4 liter ceramic flowerpots and 0.3 liter glass jars be sustainable, 7 years after official involvement has been depending on species. Copepod populations generally peaked ceased. Copepod Mesocyclops are still being used by 13 wks after introduction (August) in ceramic flowerpots and community members to keep Aedes populations at bay, and glass jars, and about 1 month later in tires, plastic buckets local transmission of dengue has been eliminated in areas and plastic flowerpots. At the time of peak abundance, where these are being used (Kay et al., 2010). average predator numbers ranged between 900 (glass jar) to > 3000 (30 liter bucket) individuals per container. Although all Predation efficiency of copepods has been reported to drop mosquito species have been eliminated from all containers, off considerably with older mosquito larvae, the effects of sometime during the 35 wks study, yet M. longisetus habitat structure and alternative prey for predation (Tietze et al., 1994). One of the characteristics that make some 104 Muhammad Sarwar: Elimination of Dengue by Control of Aedes Vector Mosquitoes (Diptera: Culicidae) Utilizing Copepods (Copepoda: Cyclopidae) cyclopoid copepods species good candidates for biological (provision of drinking water through piping water supply, use control of mosquitoes is their broad prey base. This may of tubewells and abolition of stepwells) methods that are allow some of these copepod species to survive in containers effective measures on community level (Marten, 1986). even when mosquito larvae are absent and may facilitate population establishment in new containers (Jennings et al., 1994). Thus, copepods are the group with the most potential, 4. Rearing of Copepods for and these are very cheap to yield as biolarvicides because Mosquito Control today these show a great potential to be used in the Integrated Copepod cultures are relatively easy to maintain as can be Vector Management (IVM) control programs against A. seen below, but getting the cultures started requires some aegypti worldwide. When these organisms are placed in effort and time, however, once established, copepods are able container habitats, decorative ponds and pools, these prey on to survive and reproduce well to maintain viable populations mosquito larvae, and effectively prevent mosquito (Jorge and Sheila, 2004; Suarez et al., 1992). development. The addition of copepods into large water- storage tanks has been successful in limiting dengue 4.1. Field Collection of Copepods transmission. Small copepod crustaceans of the genus Copepods for culture start-up can be collected from ponds, Mesocyclops can be removed from a pond or lake and placed ditches and other standing water sources. These can be in water containers to kill Aedes mosquito larvae. The collected by quickly dipping a suitable container (mosquito technique is simple, cheap and can be very effective. A single dipper) in the water, particularly near submerged vegetation. copepod has been observed to kill 15 to 20 first and second After dipping, slowly pour out 2/3 of the water in the instars larvae in a single day. A single introduction can container, and look at the remaining water for signs of the produce effective larval control from several weeks to several copepods, which are small, can be seen swimming with jerky years, depending on the larval habitat. movements in the collection vessel, and then quickly pour the copepods present, into a clean container. Copepods can also 3. Public Health Importance of be collected by passing water through two in-line sieves, Copepods either by pumping or by scooping water with a bucket and pouring through the strainers. The top sieve can be an There are some slight risks with introducing of copepods on a ordinary kitchen strainer to capture large debris and copepods large scale, to which scientists are still evaluating and they can be captured in the second sieve of 200 µm mesh netting should focus on their public health issues. Cyclops is covering a second strainer and secured by the edges to the intermediate host of dracunculiasis (guinea-worm disease- this strainer with a rubber band. After filtering through this set-up, is a helminth human parasite that infects peoples when they either remove the netting and invert in a container of clean ingest infected Mesocyclops in drinking water) and fish water, or turn the second sieve set-up upside down over a tapeworm (Diphyllobothrium latum ) infection. Copepods clean container and rinse with clean water. themselves are also associated with the spread of some of human diseases such as cholera, but these cannot spread it 4.2. Start-Up of Copepod Cultures Inside unless the disease is already present in the area. Another Gravid females copepod can be easily recognized because disadvantage is that these copepods are sensitive to chlorine in these carry their eggs externally on both sides of their bodies. the water (Marten and Reid, 2007). Nevertheless, despite these To start culture, set up several small containers (disposable disadvantages Mesocyclops has been sustainably used to food containers) about 3/4 full of water and introduce a almost eradicate dengue vector mosquitoes (Kay et al., 2010). single gravid female in each by picking up the female with a Under certain circumstances when copepods become public pipette and placing it in the culture container. Copepods can health problems, these can be controlled using physical be reared in pools (plastic pools), garbage cans or other (straining of water through piece of fine cloth is sufficient to suitable containers. Fill the pools with water, introduce remove cyclops and can also be killed easily by boiling at several dozen adults and monitor these containers to 60°C), chemical (chlorine destroys guinea worm larvae and determine if the copepod’s population is growing after 2-3 Cyclops in strength of 5 ppm, though this concentration gives weeks. Add approximately 1000 ml of Paramecium culture bad odor and taste to water, excess chlorine can be removed and 100 grains of wheat into each of the rearing pools. The by dechlorination, lime at dosage of 4 gm per gallon of water copepods will readily predate on the Paramecia, and the can be used, Temefos kills cyclops at concentration of 1 mg Paramecia feed on the wheat grain. Place the pools to receive per litter), biological (small fishes like Barbell and Gambusia light (natural or artificial) for at least a part of the day, but feed on cyclops to eradicate dracunculiasis) and engineering keep away from direct sun. As the wheat seed decomposes, International Journal of Bioinformatics and Biomedical Engineering Vol. 1, No. 2, 2015, pp. 101-106 105

some will start floating to the surface of the water and should during the months when mosquitoes are active. Once the be removed. Pools should be emptied and cleaned at least populations of copepod are building up in a container, these once yearly typically in late spring to avoid extreme are able to maintain viable reproducing inhabitants for temperature creating the chance of mortality. successful and excellent biological control of mosquitoes. Copepods are very useful in biological control of A. aegypti 4.3. Paramecium Cultures and A. albopictus, but also successful to manage the Plastic milk cartons (1 gallon or 1/2 gallon) work well for populations of Culex and Anopheles. These predators are culturing of Paramecium, as well as 5 gallon water jugs are most effective to predate on 1 to 4 days old mosquito larvae, also appropriate. Add 25 ml of the Paramecium caudatum but alternate food and habitat structure significantly influence stock culture to 1 gallon of spring water, then add the predation rates on prey. However, this characteristic does approximately 12 grains of wheat seed per gallon of culture not appear to negate its effectiveness as a mosquito larval and a small pinch of yeast, and wait for about two weeks. predator. The technology for culturing and field application Save about 500 ml water of each jug, refill with water and of copepods is appropriate, its costs appear to be modest and add wheat seed and yeast as before to start a new culture. little environmental risk is evident. The states should offer assistance to county members for mosquito control programs 4.4. Rearing of Copepods Outside and help to reduce their dependence on insecticides, by When copepods are cultured outside, the culture needs to be providing them a variety of natural tools to deal with covered at least 3 ft, off of the ground level. Aquaria or mosquito issues. Further tests are needed to determine the similar vessels are not ideal for copepod rearing outside. It is effectiveness of these predators in larger bodies of water such a good idea to set up several pools and to maintain additional as ditches and swales, as available data on such water cultures in smaller containers at a separate location so that holdings are scanty in tropics and subtropics. new cultures can be started in case of loss of original cultures.

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